The present invention relates to the pharmaceutical use of specific inhibitors of calcium-activated neutral proteases (CANPs). More specifically, the inhibitors may be especially used for the treatment of tumors, especially cancer. The inhibitors may be proteinaceous of character, subunits of said inhibitor proteins, or shorter peptides. The invention further relates to the manufacture of pharmaceutical preparations for the treatment of the above mentioned diseases.
Calcium-activated neutral proteases (CANPs), also known as calpain I and II, are cysteine proteases found in the cells of higher animals. They have been presumed to participate in various cellular functions mediated by calcium, but their precise function is not yet understood. Calpain I requires low concentrations of calcium to be functional (also refered to as xcexc-CANP) while calpain II requires millimolar concentrations of calcium (refered to as m-CANP). CANPs hydrolyse proteins of limited classes in vitro, including epidermal growth factor receptor, platelet derived growth factor receptor and protein kinase C. They appear to be involved in regulating the turnover and degradation of muscle myofibrillar proteins and neuronal cytoskeletal elements Murachi T., 1983, Trends Biochem. Sci., 8:167), suggesting that CANPs are involved in essential cellular functions associated with meiosis (as supported in WO 92/21373 and Logothetou-Rella H., 1994, Histol. Histopath., 9:747).
Calcium-activated neutral protease inhibitors have been used to indirectly document CANP involvement in a variety of different diseases. CANP inhibitors have been suggested for use in the treatment of myocardial infarction, ischemia and potential stroke using inhibition of platelet CANP (Puri and Colman, 1993, Blood Coagul. and Fibrin, 4:465). The degradation process of the ischemic heart is contributed to CANP (Toyo-oka et al., 1991, Japan Circulat. J., 55:1124). CANP is found in the brain of patients suffering from Alzheimer""s disease (Saito et al., 1993, Proc. Natl. Acad. Sci., 90:2628), muscular dystrophy (Hollenberg-Sher et al., 1981, Proc. Natl. Acad. Sci., 78:7742), and may play a role in the inflammatory process (Sasaki et al., 1991, Acta Biologica Hungarica, 42:231 and Logothetou-Rella H., 1994, Histol Histopath., 9:469).
One of the most lethal properties of malignant cells is their ability to infiltrate normal tissues and to metastasize to distant areas. The normal connective tissues consist of cells embedded in an extracellular matrix containing glycoproteins, collagen, elastin, and proteoglycans. There have been suggestions that tumor-associated histolytic enzymes may aid in the invasive process by removal of the matrix protein (Hart, I. et al., 1980, JNCI 64:891). Several studies have concentrated on this aspect of tumor cell biology, and increased protease production has been observed with many transformed cells (Jones, P. A. and Declerk Y. A., 1980, Cancer Res., 40:3222).
It has been reported that malignant cells in culture from human invasive urothelial carcinoma form tumor nodules and glycosaminoglycan membraneous sacs (GSG) with membrane extensions intracellularly as well as extracellularly (Logothetou-Rella H. et al., 1988, Europ. Urol. 14:61, ibid, 14:65). The same observations were made in human embryonic cell cultures Logothetou-Rella H. et al., 1989, Histol. Histopath., 4:367), while they were not found in human normal urothelial cells in culture (Logothetou-Rella H. et al., 1988, Europ. Urol., 15:259). The participation of GSG has also been reported in capillary formation which is enhanced in tumors in vivo (Logothetou-Rella H. et al., 1990, Histol Histopath., 5:55).
The characteristic extracellular matrix (GSG) of malignant and embryonic cells is PAS and PAS-diastase positive, identified by Papanicolaou stain having a light green color (EA color) and smooth to fibrillar translucent texture. GSG in malignant cells is distributed and accumulated in intracellular and extracellular membraneous sacs. The membraneous GSG sacs give rise to membrane extensions which form channels through which the green GSG is passed from the inside to the outside of the cell. This structure also enhances tumor nodule formation and invades other cells in vitro. It was further documented that this extracellular matrix (ECM) consisted of GSG bound protease resulting from cell to cell invasion (Logothetou-Rella, H., Greek patent 910100238, Mar. 6, 1991, WO92/21373, Logothetou-Rella, H. et al., 1992, Eur. Urol. 21:146, Logothetou-Rella, H., 1994, Histol Histopath., 9:243).
Nuclear vlima (NV, nuclear bullets) defines a parasitic spermatozoo-like cell produced by assymetrical, unequal cell division invading other cells (as supported by WO 92/21373 and Logothetou-Rella, H., 1993, Histol. Histopath., 8:407). NVs have been identified in malignant, embryonic, virally infected cells (Logothetou-Rella, H., 1994, Histol. Histopath., in press) and PHA-activated lymphocytes (Logothetou-Rella, H., 1994, Histol. Histopath., 9:469). Furthermore, NVs carry aneuploid sets of chromosomes, invade other host cells forming hybrids with a process similar to fertilization or viral cell infection and are sensitive to CANP inhibitors (Logothetou-Rella, H., Greek patent 910100238, Mar. 6, 1991, WO92/21373, and Logothetou-Rella, H., 1994, Histol. Histopath., 9:469, ibid 9:485, ibid 9:747).
Surprisingly, a new mechanism of cell to cell invasion subsequent to meiotic cell division and substrate (GSG bound CANP) formation, common in formation of tumors was discovered. Moreover, it was found that administering specific inhibitors of CANPs, or active subunits, thereof providing an effective concentration of said inhibitors in the human or animal body, would inhibit the aforementioned processes.